Reduced pancreatic ?-cell function and enhanced ?-cell death are key events in the pathogenesis of diabetes mellitus. In normal conditions, the pancreatic ?-cell responds to acutely elevated blood glucose with insulin secretion, and to persistently elevated blood glucose (hyperglycemia) with compensatory increase in insulin secretion and ?-cell mass. Persistent hyperglycemia may also lead to paradoxical 'glucotoxic' pancreatic ?-cell dysfunction, reduced ?-cell mass and loss of insulin content, although underlying mechanisms remain incompletely defined. Chronic hyperglycemia will lead to hyperstimulated metabolism, membrane hyperexcitability and increased [Ca2+]i. It has been suggested that hyperexcitability, high [Ca2+]i and insulin hypersecretion may play a key role in ?-cell desensitization and glucotoxicity. However, a mouse model of neonatal diabetes with genetically inexcitable ?-cells and persistently low [Ca2+]I demonstrates a marked loss of insulin content, disruption of the pancreatic architecture and loss of ?-cell mass over time. Thus, it is imperative to understand the independent contributions of hypermetabolism and hyperexcitability in ?-cell pathology of T2DM. This mouse therefore provides a diabetic model in which to examine glucotoxic effects of hyperstimulated metabolism, independent of the normally coupled hyperexcitability and high [Ca2+]i. One major goal of this proposal to address this question using in vivo and in vitro approaches in these unique animal models. Hyperglycemia induces hyperstimulated metabolism and increased reactive oxygen species (ROS) formation, which can be detrimental for ?-cells. Excessive ROS can induce mitochondrial dysfunction, and oxidative and endoplasmic reticulum (ER) stress, which are likely to be major contributors in pancreatic ?-cell glucotoxicity. A second major goal of this proposal is therefore to address the question of what is driving the gradual reduction of ?-cell mass in diabetes in vivo. I will specifically test whether the decrease in ?-cell mass in underexcitability-driven diabetes is a consequence of glucotoxicity, wheher this effect is mediated by an increase in glucose metabolism and ROS production, and whether it involves mitochondrial dysfunction. In seeking answers to these questions, the experiments proposed in this project represent a significant effort to understand mechanisms underlying diabetic glucotoxicity and will be of direct relevance to the progression of human diabetes.